Human Cervical Mucus Plugs Exhibit Insufficiencies in Antimicrobial Activity Towards Group B Streptococcus

Abstract

Preterm birth is a leading cause of neonatal mortality and lacks an effective therapy. Ascending microbial infections from the lower genital tract lead to infection of the placenta, amniotic fluid, and fetus causing preterm birth or stillbirth. Directly in the path of an ascending infection is the cervical mucus plug (CMP), a dense mucoid structure in the cervical canal with potential antimicrobial properties. In this study, we aimed to define the components of CMP responsible for antimicrobial activity against a common lower genital tract organism associated with preterm birth and stillbirths, namely, group B streptococcus (GBS). Using a quantitative proteomic approach, we identified antimicrobial factors in CMPs that were collected from healthy human pregnancies. However, we noted that the concentration of antimicrobial peptides present in the human CMPs were insufficient to directly kill GBS, and antimicrobial activity, when observed, was due to antibiotics retained in the CMPs. Despite this insufficiency, CMP proteins were able to activate leukocytes in whole blood resulting in increased rates of bacterial killing, suggesting a role for the CMP in enhancing complement-mediated killing or leukocyte activation. This study provides new insight into how the human CMP may limit ascending bacterial infection.

Figure 1.

Summary of quantitative proteomic analysis of human cervical mucus plugs (CMPs). Total proteins from 7 human CMPs were analyzed by quantitative tandem mass spectrometry. (a) Heat map representing relative abundance values of proteins from 7 CMP samples. (b) Protein relative abundance values for all consensus proteins were compared with determine proteins that were significantly enriched or depleted in CMP samples (for complete dataset, see Table S1). Thirty-five of 1201 proteins were determined to be significantly above or below the mean abundance value for all proteins (dashed line indicates mean relative abundance, Bonferroni multiple-comparison test after one-way analysis of variance [ANOVA]). (c) Principle co-ordinate analysis of protein abundance values from CMPs samples. (d) Gene Ontology analysis of biological functions of all consensus proteins identified in the CMP samples. A total of 514 of 15403 biological functions were found to be significantly over- or underrepresented in CMP samples (Bonferroni multiple-comparison test after one-way ANOVA; for complete dataset, see Table S2).

Figure 2.

Quantitative proteomic analysis reveals the immunological nature of the human cervical mucus plugs (CMPs). After complete proteomic profiling (see Figure 1), consensus CMP proteins were further analyzed for proteins that have been characterized to have immunological function. Heat maps representing relative abundance values of antimicrobial peptides (AMPs [a]), complement or complement-related proteins (b), cytokines, chemokines, or related receptors (c), and immunoglobulin or immunoglobulin-receptor proteins (d) identified in 7 human CMP samples. Boxes containing an “X” indicate that an individual protein was not identified in a given sample.

Figure 3.

Antibacterial activity of cervical mucus plugs (CMPs) against group B streptococcus (GBS) is due to antibiotics. To assess antimicrobial activity of CMPs against GBS, CMPs were sectioned, lyophilized, and resuspended in phosphate-buffered saline to a concentration of 1 mg/mL. Cervical mucus plug extracts (1 mg/mL) or erythromycin (Erm, 25 mg/mL) were spotted onto TSA plates containing GBS strains (either serotype Ia strain A909, serotype III strain COH1, or serotype V strain NCTC10/84). Antimicrobial activity was determined by the ability of the CMP extracts to prevent GBS growth at the respective spots (a and b); “Y” indicates growth inhibition and “N” indicated no growth inhibition (also see zone of clearing in b). To determine whether antibacterial activity in CMP 2 and CMP 7 could be attributed to antibiotics provided to the patient that was retained in the CMP before collection, extracts from CMP 2 and CMP 7 were treated with 1 mM penicillinase from Bacillus cereus. Growth was determined by measuring OD₆₀₀ at 4 ([c] n = 3, mean displayed ± standard error of the mean [SEM], ****P > .00001, **P > .001, Bonferroni multiple-comparison test after two-way analysis of variance [ANOVA] comparing sample to sample + penicillinase) and 24 hours ([d] n = 3, mean displayed ± SEM, ****P > .00001, Bonferroni multiple-comparison test after one-way ANOVA comparing sample to sample + penicillinase).

Figure 4.

Physiologically relevant concentrations of antimicrobial peptides (AMPs) present in cervical mucus plugs (CMPs) are insufficient to kill group B streptococcus (GBS). To assess AMP concentration in CMPs against GBS, CMPs were sectioned, lyophilized, and resuspended in phosphate-buffered saline to a concentration of 1 mg/mL. Concentrations of specific AMPs (cathelicidin, elafin, lysozyme, hNP1, and SLP1) were determined from 60 individual CMPs by enzyme-linked immunosorbent assay ([a] mean displayed). These concentrations were then used in bacterial viability assays to determine the antibacterial activity of AMPs against GBS (cathelicidin [b], elafin [c], lysozyme [d], hNP1 [e], and SLP1 [f]; n = 3, mean displayed ± standard error of the mean, *P < .05, ***P < .0005, ****P < .00005, Bonferroni multiple-comparison test after one-way analysis of variance). Higher PI/Syto9 ratio indicates lower bacterial viability, and 50% ethanol was included as a positive control. Boxes on bar graphs indicate range of detected AMP concentration in the CMPs, and arrows indicate approximate mean detected AMP concentration.

Figure 5.

Cervical mucus plug (CMP) protein enhances killing of group B streptococcus (GBS) in whole blood. To assess CMP extract enhancement of antibacterial activity against GBS in whole blood, CMPs were sectioned, lyophilized, and resuspended in phosphate-buffered saline (PBS) to a concentration of 1 mg/mL. Wild-type (WT) GBS serotype Ia (strain A909) or isogenic GBSΔcpsE were incubated with human whole blood with 25 μg/mL CMP protein or an equivalent volume of PBS. Survival index was estimated 3 hours after incubation and compares colony-forming units at 3 hours postincubation to initial inoculum (n = 5, *P < .05, **P < .005, ***P < .0005, Bonferroni multiple comparison test after one-way analysis of variance). Each assay was performed with an independent CMP and blood donor, indicated by different symbol color and connecting line. Cervical mucus plugs 2, 4, and 7 were not used in these experiments due to retained antibiotics.